On the Road towards Small-Molecule Programmed Cell Death 1 Ligand 1 Positron Emission Tomography Tracers: A Ligand-Based Drug Design Approach

PD-1/PD-L1 immune checkpoint blockade for cancer therapy showed promising results in clinical studies. Further endeavors are required to enhance patient stratification, as, at present, only a small portion of patients with PD-L1-positive tumors (as determined by PD-L1 targeted immunohistochemistry; IHC) benefit from anti-PD-1/PD-L1 immunotherapy. This can be explained by the heterogeneity of tumor lesions and the intrinsic limitation of multiple biopsies. Consequently, non-invasive in vivo quantification of PD-L1 on tumors and metastases throughout the entire body using positron emission tomography (PET) imaging holds the potential to augment patient stratification. Within the scope of this work, six new small molecules were synthesized by following a ligand-based drug design approach supported by computational docking utilizing lead structures based on the (2-methyl-[1,1′-biphenyl]-3-yl)methanol scaffold and evaluated in vitro for potential future use as PD-L1 PET tracers. The results demonstrated binding affinities in the nanomolar to micromolar range for lead structures and newly prepared molecules, respectively. Carbon-11 labeling was successfully and selectively established and optimized with very good radiochemical conversions of up to 57%. The obtained insights into the significance of polar intermolecular interactions, along with the successful radiosyntheses, could contribute substantially to the future development of small-molecule PD-L1 PET tracers.


Introduction
Immune checkpoint inhibitors and their application in immunotherapy have led to tremendous progress in oncological practice [1]. The PD-1/PD-L1 immune checkpoint works as an inhibitory regulatory mechanism to prevent excessive immune reactions and autoimmunity for self-tolerance. Cancer cells can exploit this mechanism for immune evasion by overexpressing programmed cell death 1 ligand 1 (PD-L1), thereby promoting cancer development and progression through negatively regulating T-cell-mediated immune responses and suppressing migration, proliferation and effector function of T cells, eventually inducing T cell exhaustion, a dysfunctional state of T cells lacking effector function accompanied by an increased expression of inhibitory receptors [2]. Several immune checkpoint inhibitors (ICIs) have been developed so far to block this ligand-receptor interaction, thereby reactivating the host immune system to fight cancer. Monoclonal antibodies Figure 1. The binding mechanism of lead structure 2 determined by crystallography (PDB: 5J89) using the LigandScout 4.4 software. (A) A cut-out of the crystal structure of one inhibitor molecule (orange) and two PD-L1 proteins (dark red and teal) are shown (mixed ribbon/surface representation). The pharmacophore is represented by hydrophobic features (yellow spheres), a positive ionizable area (blue spikes), a hydrogen bond donor (green vector) and a hydrogen bond acceptor (red vector). The interaction of 2 is dominated by hydrophobic interactions with both PD-L1 monomers C (red sticks) and monomer D (blue sticks). (B) The distant phenyl ring of the 2-methylbiphenyl moiety is stabilized by T-stacking interaction with the phenyl ring of the tyrosine sidechain (TYR56C) and π-alkyl interactions of methionine (MET115C) and alanine (ALA121D) sidechains. The methylphenyl ring interacts with alanine (ALA121C) and methionine (MET115D), and the methyl group of this ring fits into a pocket formed by monomer C. The two rings are twisted by about 45°. The interactions of the methoxypyridyl ring include π-π stacking with tyrosine (TYR56D) and other nonpolar interactions with monomer D. The external N-(2-amino-ethyl)acetamide unit only interacts with monomer C and seems to be more accessible for chemical modifications than the rigid inner ring system. The grid box was automatically determined by LigandScout ® and comprises around 30 × 30 × 30 Å. and two PD-L1 proteins (dark red and teal) are shown (mixed ribbon/surface representation). The pharmacophore is represented by hydrophobic features (yellow spheres), a positive ionizable area (blue spikes), a hydrogen bond donor (green vector) and a hydrogen bond acceptor (red vector). The interaction of 2 is dominated by hydrophobic interactions with both PD-L1 monomers C (red sticks) and monomer D (blue sticks). (B) The distant phenyl ring of the 2-methylbiphenyl moiety is stabilized by T-stacking interaction with the phenyl ring of the tyrosine sidechain (TYR56C) and π-alkyl interactions of methionine (MET115C) and alanine (ALA121D) sidechains. The methylphenyl ring interacts with alanine (ALA121C) and methionine (MET115D), and the methyl group of this ring fits into a pocket formed by monomer C. The two rings are twisted by about 45 • . The interactions of the methoxypyridyl ring include π-π stacking with tyrosine (TYR56D) and other nonpolar interactions with monomer D. The external N-(2-amino-ethyl)acetamide unit only interacts with monomer C and seems to be more accessible for chemical modifications than the rigid inner ring system. The grid box was automatically determined by LigandScout ® and comprises around 30 × 30 × 30 Å.

Results and Discussion
Starting from available lead structures 1 and 2, ligand docking experiments were performed against human PD-L1 (PDB: 5J89) using the LigandScout software. For validation, docking software is considered reliable when a generated pose is very similar to the original pose in the protein-ligand crystal structure reaching a root mean square deviation (RMSD) of <1.5-2 Å between the docked and original pose [15]. In our case, a RMSD of 0 Å was achieved by redocking the original ligand, highlighting its reliability. The crystal structure of hPD-L1 shows the dimeric organization of the protein units, with the lead structure(s) bound in a sandwich-like blot between two dimers with the terminal carboxylic acid or amide functionality reaching out of the hydrophobic binding pocket [12]. Based on minor interaction of the eastern part of both lead structures with the binding cleft, we started our chemical modification at the structural point with minimal target interaction. Based on compound 1, the terminal carboxylate group was equipped with a methyl (1a) or a fluoroethyl group (1b) to allow the envisioned radiolabeling with either carbon-11 or fluorine-18. Methylation of compound 2 at different positions led to the generation of a tertiary amine (2a), tertiary amide (2b) as well as a double methylated compound (2c), while fluoroethylation led to the corresponding fluoroethylated derivative (2d). Comparison of the pharmacophore revealed the loss of the interaction with ASP122C, by leaving the main pharmacophoric interactions unchanged (Supplementary Figures S2-S8). The impact of the structural changes on the docking parameters, i.e., binding affinity score and affinity, were marginal. Overall, pharmacophore assessment predicted minor effects of methylation (compound 1a, 2a, 2b and 2c) and fluoroethylation (1b, 2d) on binding affinity (Table 1). Selected small molecules derived from the lead structures were subsequently synthesized by O-and N-methylation and fluoroethylation (Scheme 1) to obtain the respective non-radioactive reference compounds for biological assessment and to set up radiosynthesis and quality control. Briefly, methyl ester 1a was obtained by Steglich esterification, conversion into an acyl imidazole and nucleophilic substitution with methyl iodide, although only the latter resulted in a sufficiently pure product (Supplementary Table S1). Fluoroethylated compound 1b was readily obtained using 2-fluoroethyl p-toluenesulfonate under basic conditions. Synthesis of 2a, 2b and 2c was straightforward using methyl iodide. No additional base was required for the synthesis of monomethylated 2a, while an additional base was needed for the formation of dimethylated compound 2c. Substance 2b was selectively produced by Boc protection of the secondary amine prior to methylation of the amide, followed by acidic Boc deprotection. Fluoroethylation in the presence of cesium carbonate resulted in 2d in good yield. In general, the conversions into methylated products were incomplete, as determined by analytical HPLC measurements, although methyl iodide was used in excess. As a result, product 1a, 2b and 2c were achieved in low overall yields of 5-8%, and 2a was isolated with a moderate yield of 41% (Scheme 1). Interestingly, when 2 was used as a substrate for fluoroethylation, no conversion occurred when N,N-diisopropylethylamine or potassium tert-butoxide was used as a base (Supplementary Table S1). The use of cesium carbonate led to carbamate formation of the amine functionality [16,17], which then functioned as a nucleophile for fluoroethylation, resulting in product 2d. Dealkylation attempts of the lead structures' aryl ethers for the synthesis of radiolabeling precursors using Lewis acids [18] (AlCl 3 , BBr 3 ) did not achieve the desired outcome. conversion occurred when N,N-diisopropylethylamine or potassium tert-butoxide was used as a base (Supplementary Table S1). The use of cesium carbonate led to carbamate formation of the amine functionality [16,17], which then functioned as a nucleophile for fluoroethylation, resulting in product 2d. Dealkylation attempts of the lead structures' aryl ethers for the synthesis of radiolabeling precursors using Lewis acids [18] (AlCl3, BBr3) did not achieve the desired outcome. Because no in-solution stability data were available, all lead structures and compounds were tested for their stability in DMSO/HEPES buffer over an extended period of time (20 days) before further in vitro analysis. In general, compounds were more stable when stored at 4-8 °C compared to room temperature. Compounds 2, 2a, 2c and 2d were highly stable in solution with marginal decomposition of less than 2% of the parent compound, while 1, 1a, 1b and 2b remained intact to over 85% within the 20-day time period (Supplementary Figures S9-S16).
Further efforts were made to establish radiolabeling strategies with respect to future structurally related compounds. Small-scale carbon-11 radiosyntheses of small molecules [ 11 C]1a, [ 11 C]2a and [ 11 C]2b were performed and peaked in a radiochemical conversion (RCC) of 49%, 54% and 57%, respectively, as determined by radio-HPLC ( Figure 2A). Because no in-solution stability data were available, all lead structures and compounds were tested for their stability in DMSO/HEPES buffer over an extended period of time (20 days) before further in vitro analysis. In general, compounds were more stable when stored at 4-8 • C compared to room temperature. Compounds 2, 2a, 2c and 2d were highly stable in solution with marginal decomposition of less than 2% of the parent compound, while 1, 1a, 1b and 2b remained intact to over 85% within the 20-day time period (Supplementary Figures S9-S16).
Further efforts were made to establish radiolabeling strategies with respect to future structurally related compounds. Small-scale carbon-11 radiosyntheses of small molecules [ 11 C]1a, [ 11 C]2a and [ 11 C]2b were performed and peaked in a radiochemical conversion (RCC) of 49%, 54% and 57%, respectively, as determined by radio-HPLC ( Figure 2A).
[ 11 C]1a was preferably produced using [ 11 C]CH 3 I in MeCN at 60 • C for 4 min ( Figures 2B and 3). As [ 11 C]2a and [ 11 C]2b originate from the same precursor molecule, the selective production of these constitutional isomers was a special challenge. [ 11 C]CH 3 I as methylating agent yielded preferably the tertiary amine (54%), and the tertiary amide was identified as the major by-product with around 20% at 150 • C and a reaction time of 4 min ( Figures 2C and 4, Supplementary Figure S18E). However, [ 11 C]2b was preferentially formed by applying the more reactive [ 11 C]CH 3 OTf synthon ( Figures 2D and 5) with only marginal amounts of the by-product [ 11 C]2a. The addition of a base, like TBAH, completely quenched the reaction. However, by-product formation was mainly a matter of an increase in radiochemical conversion rather than purity, as both products were clearly separated by HPLC. In summary, we were able to selectively radiolabel both constitutional isomers in high RCC.  ures 2C and 4, Supplementary Figure S18E). However, [ 11 C]2b was preferentially formed by applying the more reactive [ 11 C]CH3OTf synthon ( Figures 2D and 5) with only marginal amounts of the by-product [ 11 C]2a. The addition of a base, like TBAH, completely quenched the reaction. However, by-product formation was mainly a matter of an increase in radiochemical conversion rather than purity, as both products were clearly separated by HPLC. In summary, we were able to selectively radiolabel both constitutional isomers in high RCC.         Physicochemical parameters were calculated and measured for cross-validation and to establish structure-activity relationships. Measured µHPLC logP OW pH 7.4 values ranged from 3.16 to 5.02 ( Table 2). Modification of 2 (µHPLC logD 3.88) resulted in increasing lipophilicity ranked in the following order: 2b < 2a < 2d < 2c. Methylation and fluoroethylation of the carboxyl group of 1 (µHPLC logD 3.16) culminated in the highest measured lipophilicity of 4.90 and 5.02 for 1a and 1b, respectively. The calculated physicochemical parameters predicted the increase in lipophilicity in dependency of the chemical modification (Table 2). However, calculated values deviated significantly from measured values by overestimating the lipophilic character of our compounds. Given their molecular weight, high lipophilicity (µHPLC logD) and low topological polar surface area (tPSA), these compounds should easily permeate cell membranes and could potentially penetrate the blood-brain barrier [19,20]. Physicochemical parameters were calculated and measured for cross-validation and to establish structure-activity relationships. Measured µHPLC log P pH 7.4 OW values ranged from 3.16 to 5.02 ( Table 2). Modification of 2 (µHPLC logD 3.88) resulted in increasing lipophilicity ranked in the following order: 2b < 2a < 2d < 2c. Methylation and fluoroethylation of the carboxyl group of 1 (µHPLC logD 3.16) culminated in the highest measured lipophilicity of 4.90 and 5.02 for 1a and 1b, respectively. The calculated physicochemical parameters predicted the increase in lipophilicity in dependency of the chemical modification (Table 2). However, calculated values deviated significantly from measured values by overestimating the lipophilic character of our compounds. Given their molecular weight, high lipophilicity (µHPLC logD) and low topological polar surface area (tPSA), these compounds should easily permeate cell membranes and could potentially penetrate the blood-brain barrier [19,20]. hPD-L1 binding affinities of the developed compounds were assessed by means of an HTRF binding assay including the antibody atezolizumab, the macrocyclic peptide PD-1/PD-L1 Inhibitor 3 and lead structures 1 and 2 as positive controls. Binding affinities (IC 50 ) were found to be in the micro-to nanomolar range (101-9880 nM) for small molecules, while mean IC 50 values of 4.07 nM and 113 nM were determined for reference compounds atezolizumab and commercially available PD-1/PD-L1 Inhibitor 3, respectively. We were not able to reproduce the high hPD-L1 affinity values for the lead structures as published by Bristol Myers Squibb [11] (Table 3 and Supplementary Figure S17). Similar findings were recently published for the macrocyclic peptide BMS-78 [21]. This discrepancy may result from different protein concentrations applied within different assays. Affinities of our lead structure 1 (IC 50 : 202 ± 27 nM) derivatives were in the micromolar range with 1b (1440 ± 144 nM) exhibiting better binding affinity than 1a (5760 ± 613 nM). Substance 2a had the most promising IC 50 of 430 ± 62 nM, with a 4.3-fold reduced affinity compared to lead structure 2 (101 ± 10 nM). The constitutional isomer 2b showed similar hPD-L1 binding affinity to 2a (IC 50 of 524 ± 67 nM), whereas the dimethylated compound 2c demonstrated lower affinity (IC 50 of 1310 ± 185 nM). Introduction of a sterically demanding fluoroethyl carbamate moiety (compound 2d) further decreased the binding affinity to 9880 ± 1390 nM. A clear trend of measured binding affinity depending on of the sterical demand of the introduced group(s) can be drawn based on our results. The measured affinity trend was inverse for calculated binding affinity scores.
Although the 2-methylbiphenyl core of the molecules was found to be the main pharmacophore responsible for binding to hPD-L1 [12], we further deduced that (i) there is a potential underestimation of the sterical hindrance within our computational docking model, (ii) the polar residues of the molecules and associated hydrogen bonds located at the exit of the binding pocket may also have a significant impact on binding affinity and that (iii) increasing lipophilicity leads to additional non-specific binding that impedes ligand binding during the HTRF assay.
We used Spearman's rank correlation to investigate the direction and strength of our associated variables (binding affinity score, affinity and physicochemical parameters) and to statistically assess our interpretation. Non-significant weak correlations were found between calculated physicochemical parameters and measured HTRF IC 50 values (Supplementary Table S2 (1 and 2), methylated or fluoroethylated derivatives as well as the antibody atezolizumab and the peptide PD-1/PD-L1 Inhibitor 3 as positive controls using the commercially available HTRF assay. * No full dose-response curves were observed. Moreover, a set of efficiency indices was computed to assess the impact of chemical modifications as a means to optimize the selection of fragments/leads during the drug development process. These indices encompassed ligand efficiency (LE), binding efficiency index (BEI), ligand lipophilicity efficiency (LLE) and ligand-efficiency-dependent lipophilicity efficiency (LELP). These parameters were evaluated with respect to molecular size (non-hydrogen atom count), molecular weight, lipophilicity and a combination of molecular size and lipophilicity, respectively [22]. Generally, higher values of these indices indicate improved efficiency. However, it is important to note that the optimal ranges for LLE and LELP may differ based on the specific target and therapeutic field. The results obtained from our analysis (as presented in Table 4) indicate that the reduction in binding affinity was not compensated by the concurrent increase in molecular size (LE) or molecular weight (BEI). Additionally, the elevation in lipophilicity pushed the compounds into a domain associated with potentially unfavorable pharmacokinetic characteristics (LEE, LELP). Table 4. Efficiency indices used to guide drug development. LE = ligand efficiency, BEI = binding efficiency index, LLE = ligand lipophilicity efficiency, LELP = ligand-efficiency-dependent lipophilicity efficiency. Indices were calculated as described before [22,23]

General Information
All solvents and chemicals were obtained from commercial suppliers and used without further purification unless otherwise stated.
Setup 3: An isocratic mixture of 60% A: 40% B was used as mobile phase. For semi-preparative purification, an Agilent 1200 series LC system was paired with a SUPELCOSIL™ ABZ+ HPLC column, 5 µm, 25 cm × 10 mm (Merck KGaA, Darmstadt, Germany). Solvent "A" consisted of 90% v/v MeCN plus 10% v/v Milli-Q H 2 O and solvent "B" of 10 mM sodium phosphate buffer adjusted to pH 7.4 with 1 mol/L NaOH. The flow rate was set to 5 mL/min. Setup 4: A mobile phase gradient of 50% A: 50% B to 75% A: 25% B within 10 min and a hold until the end of the run was used.
Setup 5: An isocratic mobile phase of 75% A: 25% B was used. Setup 6: A mobile phase gradient of 50% A: 50% B to 60% A: 40% B within 10 min and a hold until the end of the run was used.
Setup 7: An isocratic mobile phase of 60% A: 40% B was used. For high-performance liquid chromatography measurements after radiosynthesis, an Agilent Technologies 1620 Infinity system was utilized with an Aqua ® C18, 5 µm, 125 Å, LC column 150 × 4.6 mm (Phenomenex Inc., Aschaffenburg, Germany) as stationary phase and GINA Star Software for data acquisition.
Setup 8: A mobile phase of 80% A: 20% B and a flow rate of 1.0 mL/min was used. "A" consisted of 90% v/v MeCN in Milli-Q H2O and "B" of 10 mM sodium phosphate buffer pH 7.4 with 1 mol/L NaOH. For biocide purposes, a spatula tip NaN3 (Merck KGaA, Darmstadt, Germany) was added to "B". Setup 9: An isocratic mobile phase of 50% A: 50% B and a flow rate of 1.0 mL/min was used. "A" consisted of 90% v/v MeCN in Milli-Q H2O and "B" of 50 mM ammonium dihydrogen phosphate (Honeywell International Inc., Charlotte, NC, USA) adjusted to pH 9.3 with 5 mol/L NaOH. For biocide purposes, a spatula tip's worth of NaN3 was added to "B" and eventually filtered through a pleated filter (Cytiva, Marlborough, MA, USA).
Setup 10: For logD measurements, an Agilent 1200 series was paired with an Agilent 1100 autosampler and Agilent 1100 UV detector, an apHera™ column (10 × 6 mm, 5 µm; Merck KGaA, Darmstadt, Germany), GINA Star Software for data acquisition and a mobile phase gradient of 10% A and 90% B to 100% A within 9.4 min and back to starting conditions until minute 12. An equilibration time of 2 min before measurements was set. Solvent "A" consisted of methanol (Merck KGaA, Darmstadt, Germany) and solvent "B" of 10 mM sodium phosphate buffer pH 7.4. The flow rate was set to 1.5 mL/min. Setup 11: For semi-preparative purification after radiosynthesis, the GE TRACER-lab™ FX2 C synthesis module (General Electric Medical Systems, Uppsala, Sweden) was paired with a Sykam S1122 pump (Sykam, Eresing, Germany), a BlueShadow UV detector (KNAUER Wissenschaftliche Geräte GmbH, Berlin, Germany) and a SUPELCOSIL™ ABZ+ HPLC column, 5 µm, 25 cm × 10 mm. The solvent consisted of 55% MeCN and 45% 10 mM sodium phosphate buffer adjusted to pH 7.4 with 1 mol/L NaOH. The flow rate was set to 5 mL/min.

Ligand Docking Experiments
Compound structures were protonated to pH 7.4 using MarvinSketch 22.13 software. Ligand docking was then performed with LigandScout 4.4 software (Inte:Ligand GmbH, Vienna, Austria) using the AutoDock Vina 1.1 program and PDB code 5J89 (PD-L1 monomer C and D). Water and ethylene glycol molecules were removed prior to docking. Docking was performed in triplicates for more consistent results using the default settings (Exhaustiveness: 8; max. number of modes: 9; max. energy difference: 3).

In-Solution Stability Measurements
For stability measurements of lead structures 1 and 2 as well as substances 1a, 1b, 2a, 2b, 2c and 2d, each compound was dissolved separately in 1:1 DMSO/HEPES buffer. The buffer contained 10 mM HEPES (Merck KGaA, Darmstadt, Germany) adjusted to pH 7.5 with NaOH and 150 mM NaCl. The solutions were stored in an amber glass vial (Thermo Fisher Scientific Inc., Waltham, MA, USA) at room temperature (24 • C) or in the fridge (0 • C) and analyzed over a period of 3 weeks via HPLC, measuring three technical replicates. HPLC setup 1 was used for 1a and 1b and HPLC setup 3 for 1, 2, 2a, 2b, 2c and 2d. Detection was performed at 216 nm. The area under the curve (AUC) was used as the evaluation parameter and plotted over time.

Lipophilicity and Calculated Physicochemical Properties
The measurements of lipophilicity of precursors and products were performed according to the HPLC method of Donovan and Pescatore [24] and Vraka et al. [25]. An internal standard mixture consisting of 1% v/v toluene (Merck KGaA, Darmstadt, Germany) and 0.438 mmol/L triphenylene (Merck KGaA, Darmstadt, Germany) in methanol was added to sample solutions of approx. 1 mg/mL dissolved in DMSO.
After separation by HPLC setup 10 and determination of retention times by simultaneous detection at 254 and 280 nm in three technical replicates, the calculation of log P pH 7.4 OW (logD) was performed as described before [25]. Three logP values of the reference substances were taken from the literature, resulting in a mean logP value of the analyte (µHPLC log P

Binding Affinity Measurements
A commercially available homogeneous time-resolved fluorescence (HTRF) PD-1/PD-L1 Binding Assay Kit (Cisbio Bioassays SAS, Codolet, France, part no. 64PD1PEG) was used to determine in vitro binding affinities towards human PD-L1. The assay was prepared and performed according to the binding assay kit protocol using white, flat-bottom, low-volume Greiner 384-well plates (Merck KGaA, Darmstadt, Germany) and an HTRF-compatible Flexstation 3 Multi-Mode Microplate Reader (Molecular Devices LLC., San Jose, CA, USA) for read-out. Tenfold dilution series of the small-molecule compounds were prepared at a constant final DMSO concentration of 0.2%, as it is recommended to keep DMSO below 0.5% (Supplementary Figure S1). A threefold dilution series without DMSO was used for the antibody atezolizumab (MedChemExpress, Monmouth Junction, NJ, USA) and a tenfold dilution series without DMSO for the peptide PD-L1/PD-1 Inhibitor 3 (Selleck Chemicals Llc, Houston, TX, USA). Assay validation was monitored using the provided PD-1/PD-L1 antibody from the assay kit. Experiments were repeated for a total of three times. IC 50 calculation was performed with GraphPad Prism 8 (GraphPad Software, Inc., Boston, MA, USA) using the variable slope (four parameters) dose-response fit. Data normalization was performed for inter-assay comparison of multiple experiments according to the procedure advised by Cisbio. The ratio between the detected wavelengths was calculated (Formula (1)), and the background fluorescence signal was subtracted from the ratio to obtain the delta ratio (∆R) (Formula (2)) from which delta F (∆F), which reflects the signal to background ratio of the assay, can be calculated (Formula (3)). For normalization, ∆F/∆F max was calculated (Formula (4)) and plotted against the sample concentration on a logarithmic scale. Data normalization had no impact on IC 50 calculation.

Radiosyntheses with Carbon-11
Small scale radiosyntheses were performed using a GE TRACERlab™ FX2 C module (General Electric Medical Systems, Uppsala, Sweden). Radionuclide production and production of [ 11 C]methylating agents was performed as described before [26]. In short, [ 11 C]CO 2 was produced in a GE PETtrace cyclotron (General Electric Medical Systems, Uppsala, Sweden) by irradiation of a gas target containing N 2 and 0.5% O 2 using the 14 N(p,α) 11 C nuclear reaction with up to 16.5 MeV protons. [ 11 C]CO 2 was reduced to [ 11 C]CH 4 by H 2 gas and nano-powdered nickel as a catalyst at 400 • C. [ 11 C]CH 4 was converted into [ 11 C]CH 3 I with I 2 at 720-740 • C by a radical reaction. Subsequently, [ 11 C]CH 3 I was trapped in the solvent (i.e., MeCN or DMSO). Alternatively, [ 11 C]CH 3 I was passed through a silver triflate containing column at 200 • C for [ 11 C]CH 3 OTf production, which was then used as a methylation reagent. A total of 100 µL of the [ 11 C]CH 3 I or [ 11 C]CH 3 OTf containing solution was added to the precursor solution.
For the radiosynthesis of [ 11 C]2a and [ 11 C]2b, 100 µL [ 11 C]CH 3 I solution was added to 0.5 or 1 mg of compound 2 dissolved in 400 µL MeCN or DMSO and stirred at room temperature, 60 • C, 100 • C or 150 • C for 2 or 4 min. Using [ 11 C]CH 3 OTf, reactions were conducted at 150 • C for 4 min with and without 1 eq. TBAH as a base. MeCN was used for radiolabeling at room temperature and 60 • C, whereas DMSO was used for reactions at 100 • C and 150 • C.
Eventually, the reactions were quenched with 100 µL H 2 O, and the radiochemical conversion (RCC) was determined by HPLC using setup 8 and setup 9 for [ 11 C]1a and [ 11 C]2a, respectively.
For further in vitro evaluation, [ 11 C]2a was purified by semi-prep. HPLC (HPLC setup 11). Organic solvent was removed by solid-phase extraction: The product fraction was diluted with 40 mL H 2 O, transferred onto a Sep-Pak C18 Plus Short cartridge (Waters Corporation, Germany), washed with 10 mL H 2 O and eluted with 1.5 mL ethanol followed by 5 mL saline. Radiochemical purity was determined by HPLC setup 9.

Statistical Analysis
Values are depicted as mean ± standard deviation (SD), and experiments were performed in triplicates and repeated at least three times. Peak areas in the radioactivity channel were corrected for decay during HPLC measurements, and radiochemical conversion was calculated according to Equation (5).

Conclusions
A total of six small molecules, derived from a ligand-based drug design strategy, were successfully synthesized and comprehensively characterized for their physicochemical properties, stability and binding affinity towards human programmed death-ligand 1 (hPD-L1). Employing an extensive small-scale radiolabeling investigation, we achieved selective labeling of constitutional isomers, wherein the carbon-11 label was introduced selectively onto either an amine ([ 11 C]2a) or an amide functionality ([ 11 C]2b) with remarkable radiochemical conversion (RCC) exceeding 50%. Although the pursuit of small-molecule ligands for hPD-L1 with sufficiently high affinities for PET imaging applications remains a challenge, this study significantly expanded our understanding of the influence of structural modifications of compounds based on the (2-methyl-[1,1 -biphenyl]-3-yl)methanol core scaffold and their monoselective carbon-11 N-methylation of amides in the presence of amines.